ABSTRACT: ?Developing a high-throughput method to validate microRNA biogenesis in vivo? microRNAs (miRNAs) are a family of small regulatory RNAs involved in repression of gene expression. They recognize their target mRNAs though base pairing and upon binding, trigger translational repression, deadenylation and decay of the target mRNAs. Given that each miRNA has the potential to regulate hundreds of genes, and several dozens of miRNAs are expressed in each cell type, miRNAs are one of the master regulators of gene expression at post-transcriptional level in animals, plants and virus. Thus, the elucidation of the miRNA complement is essential to understand gene regulation and mRNA turnover. The goal of this project is to develop a high-throughput method for miRNA discovery and experimental validation in vivo. Current miRNA discovery methods heavily rely on providing sequencing evidence of a small RNA that fits into a hairpin-like structure predicted bioinformatically. The problem is that sample availability or the abundance of a specific cell type in a tissue may be limited and hamper the number of miRNAs that can be backed-up with sequencing support. In these situations, the current solution is to sequence deeper, increasing overall costs. On the other hand, the presence of a small RNA of ~22-nt does not guarantee the belonging to the miRNA family, as random degradation of cellular RNAs also will produce small RNAs of this size and only tedious experimental validation of their processing into intermediate species and mature miRNA can confirm their identity. A recent curation work found that of the over ~7,000 metazoan miRNAs in miRBase, the reference miRNA database, only 1,175 fulfilled the features necessary to pass as miRNAs, directly rejected 3,470 as false positives and lacked enough sequencing evidence to call another 2,105 miRNAs. Therefore, there is an unmet technological need to develop a miRNA validation method that boosts the access to the miRNA candidates even from limited samples and that allows rigorous multiplexed experimental validation of their processing. With the synergy of an interdisciplinary team that combines expertise in microRNAs and zebrafish manipulation (Dr. Cifuentes, PI) and bioinformatics (Dr. Moxon, co-I), we will develop miRAGe (miRNA Analysis Genome wide), a high-throughput method to integrate miRNA prediction and their experimental validation in vivo. The fundamental concept behind miRAGe is the use of living cells as ?organic computers? to analyze miRNA processing. We will use massively parallel oligo pool synthesis to prepare the miRNA candidate precursors and test their processing in injected zebrafish embryos. Small RNA sequencing will determine which candidates are true microRNA by detecting their ~22-nucleotide mature product and their stereotypic intermediates. Overall, the results from this project will transform the access of the scientific community to high quality, experimentally validated miRNA data from any organism with genomic data available, thus enhancing future transversal studies on post-transcriptional regulation.